Osteoarthritis (hereinafter also referred to “OA”) is one of so-called degenerative diseases which occur on the basis of aging. The number of OA patients has been steadily increasing in the current aging society, but no adequate diagnostic or therapeutic method therefor has yet been established. The initial pathologic changes in OA are thought to be the degeneration and wear of the joint cartilage caused by mechanical stress due to aging. These changes advance at an extremely slow rate and lead to the gradual progression of pain.
The current drug therapy of OA uses, in systemic therapy, 1) an antipyretic analgesic (acetaminophen) or 2) nonsteroidal anti-inflammatory drugs (hereinafter also referred to as NSAIDs), or, in topical therapy (intra-articular injection), 3) hyaluronic acid (hereinafter also referred to as HA) preparations, and 4) steroid preparations. When systemic drug therapy including NSAIDs does not alleviate the pain or swelling of a local part of a joint, the intra-articular injection of steroid preparations, which have the most excellent anti-inflammatory activity, has been heretofore carried out. However, steroid preparations have problems in terms of safety e.g. because they may cause intra-articular injection syndrome (steroid arthropathy) and may have systemic side effects. Thus, HA preparations are becoming more useful as a safer intra-articular injection alternative to steroid preparations.
HA is an endogenous polysaccharide composed of repeating units of N-acetylglucosamine and glucuronic acid. HA serves to hold the viscoelasticity and load-absorbing and lubricating effects of synovial fluid as a main component constituting the synovial fluid, and, in the cartilage matrix, plays a central role in maintaining the water-holding capacity and viscoelasticity thereof by binding to cartilage proteoglycan to form a polymer called aggrecan.
Since the injection of HA with a molecular weight of about 600,000 daltons or a crosslinked product thereof into a knee joint eliminates pain derived from OA, HA preparations are widely used as one of OA therapies. Further, a high molecular weight type HA preparation having a molecular weight close to that of HA present in normal synovial fluid (product name: Suvenyl™, manufacture and distribution: Chugai Pharmaceutical Co., Ltd.) is approved in Japan with regard to an indication for the elimination of knee pain associated with rheumatoid arthritis (hereinafter also referred to as RA). In this regard, it is said that the molecular weight of HA correlates with the potency thereof and the effect of a high molecular weight type HA is longer-lasting and more potent than that of a low molecular weight type HA.
It is generally thought that HA preparations reverse the impaired viscosity and elasticity of synovial fluid resulting from the pathologic condition of OA (or RA), to eliminate pain. However, an effect of an externally added HA preparation lasts over a long period of time whereas the HA disappears from synovial fluid within several days. Thus, there is also suggested a possibility that an externally added HA preparation could act in eliminating pain by a mechanism different from that for the above-described improvement in the viscoelasticity of synovial fluid. Examples of the mechanism include an inhibitory effect against OA synovitis which is to be described hereinafter.
The pathogenic mechanism for pain and inflammation in OA still has many unclear points, but attention has been recently given to a possible link of the mechanism with synovitis which is secondarily triggered by cartilage degeneration. OA synovitis is thought to be the major exacerbation factor promoting the pathologic condition of OA because it not only becomes the main cause of symptoms of pain and inflammation such as hydrarthrosis or heat, but also accelerates joint destruction through the production of proteases, cytokines, and radicals. In addition, OA synovitis does not exhibit such a significant proliferative change as seen in RA, but has many aspects common to RA synovitis, such as synovial cell proliferation, angiogenesis, hyperemia, and subsynovial edema and fibrosis. Thus, the control of OA synovitis is important from the standpoint of efficiently eliminating pain and inflammation in OA to prevent the progression of the pathologic condition thereof.
The effect of HA on the synovial membrane has not been fully elucidated yet, but it is known from an experiment using an isotope that HA accumulates and is present over a longer period of time in the synovial membrane than in the articular cavity. It has been also reported that receptors recognizing HA (CD44 and RHAMM (receptor for HA-mediated motility)) are present on the surface layer of synovial cells constructing synovial tissue and that synovial cells are provided with a mechanism for incorporating HA even with a molecular weight of 2,000,000 or more into the cell through CD44 on the surface layer thereof. It is suggested from these findings that at least part of the pain-eliminating effect of HA is exerted through its effect on the synovial membrane; however, HA preparations do not have enough effect to inhibit inflammatory symptoms per se induced in OA synovitis, and therefore, their effect against OA and RA, which exhibit strong inflammatory symptoms, are far from adequate.
As drugs controlling synovitis have been well known a class of drugs called disease modifying anti-rheumatic drugs (hereinafter also referred to as DMARD) used in treating RA. Among others, methotrexate (hereinafter also referred to as MTX) is a drug having advantages, for example, of having excellent potency and of having relatively short time before the exertion of its effect. However, MTX is known to cause, in regions other than joint which is to be treated, serious side effects (hepatopathy, hematopoietic disorder, lung disorder, gastrointestinal tract disturbance, etc.) ascribed to the mechanism of the action of MTX, because the use of MTX has been approved only for systemic administration (only capsules of MTX have been currently approved as a pharmaceutical for treating RA in Japan; tablets and injections thereof have been approved abroad). As a result, it is essential in the use of MTX that side effects are sufficiently monitored and a measure for the incidence of side effects be taken. Because of the great fears of such side effects, synovitis-suppressing drugs including MTX have no approved indication for other joint diseases such as OA, whose symptoms are milder than those of RA. Thus, if a means for lessening the systemic side effects of MTX or a means for enabling MTX to exert its action only in the region where the development of the beneficial effect of MTX is required is found out, it will become possible not only to provide a safer RA therapy, but also to use MTX in a wide range of joint diseases.
Several methods for localizing the effect of MTX only to the inside of a joint and the synovial membrane have been attempted as means for lessening the side effects of MTX and extracting only the desired effect thereof. For example, a method for topically (intra-articularly) administering MTX alone has been reported; however, it does not enable the sufficient beneficial effect thereof to be exerted, because MTX rapidly disappears from the joint cavity. A method for using MTX formed into a liposome to improve the intra-articular retention thereof utilizing the phagocytic capacity of macrophage has been also reported; however, its clinical usefulness has not yet been confirmed. Thus, technical improvement is still necessary in order to lessen the side effects of MTX as a therapeutic drug for joint diseases and extract only the expected effect.
As described above, the synovial membrane is a tissue in which HA is apt to accumulate. In addition, the synovial cell is provided with a mechanism for incorporating HA into the cell through an HA receptor such as CD44. Thus, HA seems to have a possibility of providing a carrier for accumulating a drug in the synovial membrane. Several techniques using HA as an internal carrier for drugs have been previously reported. However, there are few known examples of applying HA to a technique with regard to the creation of a drug delivery system (hereinafter also referred to as DDS) which is for therapeutic drugs suitable for joint diseases, particularly drugs suitable for controlling synovitis, represented by MTX.
Previously known examples of reports include a polysaccharide-drug conjugate, in which a drug is conjugated with a polysaccharide including HA through a peptide chain (Patent Document 1: Japanese Patent Laid-Open No. 05-39306, Patent Document 2: International Publication WO94/19376, and so on). Each of the documents relates to a DDS technique for anticancer drugs, and states that the DDS technique improves the transfer of the drug into a cancer tissue.
In Japanese Patent Laid-Open No. 05-39306, MTX is used, intended as an anticancer drug. However, since the technique is characterized by the improved transfer of MTX into a cancer tissue and the absence of long-term persistence thereof in the body, the binding rate of MTX is made high (6.4 to 19% in a working example of the patent document) and the molecular weight of HA is made low (100,000 daltons in a working example in the patent document), in order to enhance the anticancer effect. In addition, binding of a peptide chain with the hydroxy group of HA through isourea bonding makes the conjugate less stable in an aqueous solution.
There are also examples of reports, in each of which a conjugate in which HA is conjugated with a drug has been used as a therapeutic drug for joint diseases. For example, International Publication WO99/59603 (Patent Document 3) discloses a conjugate in which HA is conjugated with a drug through a spacer such as a butyleneamine group (—C4H8NH—) or an octyleneamine group (—C8H16NH—). This patent document describes the conjugate as that can exert a beneficial effect in the state where the drug is kept conjugated, assuming the beneficial effect outside cells. In this conjugate, in fact, the conjugation between a drug and HA through the spacer is relatively strong, and therefore this technique is difficult to apply to a drug which, like MTX, can not exert a beneficial effect unless it is released from the conjugate.
In addition, this patent document is directed to a conjugate using a matrix metalloproteinase inhibitor (hereinafter also referred to as MMPI) as a drug, and the disclosed working examples also relate only to a MMPI conjugate. No conjugate using MTX as a drug is specifically disclosed, and no description of the usefulness of the conjugate as a pharmaceutical is also contained.
International Publication WO02/44218 (Patent Document 4) discloses an HA-drug conjugate produced by using a spacer in which a particular group (norbornene) is further bound to a 13-amino-4,7,10-trioxatridecanyl group and forming carbamate bonding between the norbornene and the hydroxy group of HA. However, this conjugate also seems to be intended to show a beneficial effect outside cells as in Patent Document 2; the effect is exerted in the state where the drug is kept conjugated. Thus, this technique is difficult to apply to a drug such as MTX which can not exert a beneficial effect unless it is released from the conjugate. In addition, Patent Document 3 is directed to a conjugate using MMPI as a drug, and no indication of a conjugate using MTX as a drug is given.
As discussed previously, none of the above-mentioned documents describes an HA-MTX conjugate using MTX, and neither description nor indication of the use of an HA-MTX conjugate as a therapeutic drug for joint diseases is given.
In addition, the present inventors have demonstrated that, in a method for synthesizing an HA-drug conjugate known as a prior art, the molecular weight of HA greatly decreases during synthesis process, leading to the loss of the beneficial effect of HA. A conventional method for synthesizing an HA-drug conjugate uses general conditions of organic synthetic reaction and after-treatment, but the method is necessary to be further improved in order to prepare a conjugate of high molecular weight HA and a drug.
As described above, an HA-drug conjugate used as a pharmaceutical, particularly a high molecular weight HA-drug conjugate suitable for treating joint diseases, a preparation using the same, and a method for synthesizing the conjugate have not been previously known.                Patent Document 1: Japanese Patent Laid-Open No. 5-39306        Patent Document 2: International Publication WO94/19376 pamphlet        Patent Document 3: International Publication WO99/59603 pamphlet        Patent Document 4: International Publication WO02/44218 pamphlet        